Stroke remains a leading cause of human death and disability while very few effective treatments are available for stroke patients. Stem cell transplantation therapy provides the possibility to regenerate and repair damaged brain tissues after ischemic stroke. The investigation takes a comprehensive and unprecedented approach to promote both trophic supports as well as cell replacement potential of pluripotent stem cells to develop a highly effective stem cell therapy for ischemic stroke. We propose that enhancing the survival and regenerative properties of transplanted cells as well as an improved host environment are critical for a successful stem cell stroke therapy. To reach this goal, our previous and preliminary studies have demonstrated a marked protective effect and increased functional benefits of combining hypoxia preconditioning (HP) and other regenerative strategies including optogenetic techniques and up regulated multiple trophic factors in the ischemic brain promoted by peripheral stimulation. Our central hypothesis is that a combination strategy of HP-primed NPCs subjected to optogenetic manipulations and improved host environment will allow better survival of transplanted as well as endogenous cells, enhance neurogenesis/angiogenesis via both exogenous and endogenous mechanisms, and results in optimal tissue repair and functional recovery after stroke. In neural progenitor cells (NPCs) derived from mouse induced pluripotent stem (iPS) cells, we will express the blue light- sensitive channelrhodopsin (ChR) channels and test the possibility that activation of ChRs by blue light stimuli or by the luciferase/ChR proten (luminopsis) substrate coelenterazine (CTZ) is a feasible and effective method to improve and evaluate neuronal differentiation, integration into host neural networks and neuronal connections after transplantation into the ischemic brain. We will examine the strategies to promote tissue repair and provide evidence for the morphological and functional restoration of ischemic brain structures in the unique barrel cortex ischemic stroke model of mice. We will demonstrate the feasibility and benefits of expression/activation of ChR channels in iPS-NPCs in vitro (Specific Aim 1) and after implantation into the post-ischemic barrel cortex (Aim 2). Based on the well-defined whisker- thalamus-barrel cortex pathway, structural and functional restoration of disrupted whisker-barrel activities will be evaluated usin a combination of cell specific and neuronal pathway specific measurements, including optogenetic, electrophysiological and optical imaging recordings (Aim 3). The proposal is from three research laboratories with complementary expertise in biophysics, electrophysiology, cellular/molecular biology and clinical neurosciences. The demonstration of cellular and tissue repairing benefits of iPS-NPCs in a particular brain structure is critical for the development of mechanism based cell transplantation therapy and the strategies will have great impacts on pre-clinical and clinical studies.